PURIFICATION AND CATION-BINDING PROPERTIES OF THE RECOMBINANT HUMAN S100 CALCIUM-BINDING PROTEIN A3, AN EF-HAND MOTIF PROTEIN WITH HIGH-AFFINITY FOR ZINC

The calcium-binding protein S100A3 is an unusual member of the S100 fa mily, characterized by its very high content of Cys. In order to study the biochemical, cation-binding, and conformational properties, we pr oduced and purified the recombinant human protein in Escherichia coli. The recombinant protein forms noncovalent homodimers, tetramers, and polymers in vitro with a subunit molecular weight of 11,712. The Zn2+- binding parameters of S100A3 were studied by equilibrium gel filtrati on and yielded a stoichiometry of four Zn2+ per monomer with a [Zn2+]( 0.5) of 11 mu M and a Hill coefficient of 1.4 at physiological ionic s trength. The affinity for Ca2+ is too low to be determined by direct m ethods (K-Ca > 10 mM). Ca2+- and Zn2+-binding can be followed by optic al methods: the Trp-45 fluorescence is high in the metal-free form and addition of Zn2+ and Ca2+, but not of Mg2+, leads to a 4-fold quenchi ng. Ca2+ and Zn2+ promote also quite similar conformational changes in the Tyr and Trp environment as monitored by difference spectrophotome try. Fluorescence titrations with Zn2+ confirmed that there is one set of high affinity binding sites with a [Zn2+](0.5) of 8 mu M and a Hil l coefficient of 1.3. Binding of Zn2+ to a second set of low affinity sites induces protein precipitation. Fluorescence titrations with Ca2 confirmed the very low affinity of S100A3 for this ion with a [Ca2+]( 0.5) of 30 mM and slight negative cooperativity. Mg2+ has no effect on this binding curve. Of the 10 Cys residues in S100A3, 5 only are free thiols, and accessible to 5,5'-dithiobis(2-nitrobenzoic acid); they d isplay a high reactivity in the metal-free and Ca2+ form, but a 20-fol d lowered reactivity in the Zn2+ form of S100A3. Ca2+-binding promotes the formation of a solvent-accessible hydrophobic surface as monitore d by the 60-fold fluorescence increase of 2-p- toluidinylnaphthalene-6 -sulfonate, whereas Zn2+ has no noticeable influence. Our data indicat e that Ca2+ and Zn2+ do not bind to the same sites and that under phys iological conditions S100A3 is a Zn2+-binding rather than a Ca2+-bindi ng protein; nevertheless, very specific conformational changes are int roduced by either Ca2+ or Zn2+. Since no Zn2+ binding motif of known s tructure was identified in the primary sequence of S100A3, the results are suggestive for a novel Zn2+-binding motif.